Progesterone-induced sphingosine kinase-1 expression in the rat uterus during pregnancy, and
Yow-Jiun Jeng, Victor R. Suarez, Michael G Izban, Hui-Qun Wang, Melvyn S. Soloff
Departments of Obstetrics and Gynecology, Pathology, University of Texas Medical Branch,
Galveston, Texas 77555-1062
Short title: Sphingosine kinase
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Articles in PresS. Am J Physiol Endocrinol Metab (December 12, 2006). doi:10.1152/ajpendo.00373.2006
Copyright © 2006 by the American Physiological Society.
Sphingosine-1-phosphate (Sph-1-P), a product of sphingomyelin metabolism, can act via a
family of cognate G-protein-coupled receptors, or as an intracellular second messenger for
agonists acting through their membrane receptors. In view of the general growth promoting and
developmental effects of Sph-1-P on target cells, we hypothesized that it plays a role in
adaptation of the uterus to pregnancy. We analyzed its potential role, and that of the related
lysophospholipid, lysophosphatidic acid, in the pregnant rat uterus by examining changes in
mRNA levels of cognate receptors and enzymes involved in their turnover. Of these, only
sphingosine kinase 1 (SphK1) was markedly changed (about a 30-fold increase), being
localized in the glandular epithelium, vasculature and the myometrium. Uterine SphK1 mRNA
and protein levels paralleled those of serum progesterone, and treatment with progesterone or
an antagonist elevated or reduced SphK1 mRNA expression, respectively. Progesterone also
increased SphK1 mRNA steady state levels in a rat myometrial/leiomyoma cell line (ELT3).
Overexpressing human SphK1 in these cells resulted in increased levels of the cell cycle
regulator, cyclin D1, and increased myosin light chain phosphorylation. Ectopic expression of
SphK1 also resulted in increased proliferation rates, possibly in conjunction with increased
cyclin D1 expression. These studies suggest that the uterine expression of SphK1 mediates
processes involved in growth and differentiation of uterine tissues during pregnancy.
Keywords: ELT3 cells; myosin light chain phosphorylation; cyclin D1, lysophospholipids;
sphingosine-1-phosphate; sphingosine-1-phosphate lyase
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The bioactive lysophospholipids, sphingosine-1-phosphate (Sph-1-P)1and lysophosphatidic
acid (LPA) are growth factors that act through a family of G protein coupled receptors (GPCRs),
S1P1through S1P5and LPA1through LPA4, respectively (25), causing a broad array of effects
on target cells including cell proliferation, survival, migration, adhesion molecule expression,
and morphogenesis (26, 31, 44, 47, 52). Sph-1-P also plays a role in vasculogenesis in the
mouse embryo (4, 27). Both lysophospholipids increase myosin light chain phosphorylation in
platelets and endothelial cells (10; 37) by inhibiting myosin light chain phosphatase (43). They
also stimulate DNA synthesis in human myometrial cells in primary culture (20, 32). Platelets
are the major source of circulating Sph-1-P and LPA, releasing the lysophospholipids in
response to prothrombotic stimuli (47, 54). Circulating Sph-1-P also arises by constitutive
secretion by cells of hemangioblastic lineage, such as monocytes, and by mast, endothelial, and
red blood cells (54).
Intracellular Sph-1-P is synthesized by a variety of cell types, and acts as a second messenger
for a growing list of activated membrane receptors, such as platelet derived growth factor (34),
muscarinergic acetylcholine (29), purinergic P2Y2(2), tumor necrosis factor-α (53), epidermal
growth factor (30), formyl peptide (1), bradykinin B2 (5), and specific antigen (9, 28) receptors.
Agonist-induced activation of these receptors results in the activation of sphingosine kinase
(SphK), which catalyzes the rapid and transient phosphorylation of sphingosine to Sph-1-P. Two
isoforms of SphK, which are encoded by separate genes, have been characterized (44).
1Abbreviations used: GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; LPP1,
phosphohydrolase-I; LYP2, lysophospholipase-2; MLC20, myosin light chain 20; RT-PCR; reverse
transcription polymerase chain reaction; Sph-1-P, sphingosine-1-phosphate; SphK1, sphingosine kinase-
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Comparison of the amino-acid sequences of the two indicates that human SphK1 lacks
hydrophobic transmembrane regions, whereas human SphK2 appears to be a membrane
protein with four transmembrane regions. The two enzymes differ in size, tissue distribution,
developmental expression, substrate specificity, specific activity, and likely cellular roles [see
(46) for references]. The turnover of Sph-1-P is mediated either by dephosphorylation by
specific Sph-1-P phosphohydrolases or by cleavage to ethanolamine phosphate and
hexadecenal by a pyridoxal-dependent Sph-1-P lyase.
Extracellular LPA is synthesized by platelets and other cell types by the phospholipase A-
catalyzed deacylation of phosphatic acid, or from lysophosphatidylcholine in a reaction
catalyzed by a specific phosphodiesterase, lysophospholipase D (autotaxin) (35, 49, 50). LPA is
degraded through the actions of intracellular phosphatases such as phosphohydrolase-I (LPP1)
and lysophospholipase-2 (LYP2) (7, 35).
In considering the broad functions of Sph-1-P, it is surprising that disruption of the SphK1 gene
in mice was found to cause no obvious abnormalities in fertility (3). There was a significant
decrease in serum Sph-1-P levels in these mice, but Sph-1-P concentrations in most tissues
were not markedly reduced. These findings indicate that additional SphKs, such as SphK2,
might compensate for the ablation of SphK1 expression. It is clear that Sph-1-P is vital for
developmental processes, as disruption of the S1P1gene in mice caused embryonic
hemorrhage leading to intrauterine death between E12.5 and E14.5 (27). Vascular maturation
was incomplete in these embryos due to a deficiency in vascular smooth muscle cells and
pericytes that normally would support endothelial tube structures (27). Disruption of S1P2and
S1P3together also results in marked perinatal lethality (19).
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The rat myometrium undergoes increased cellular division during the first half of pregnancy,
followed by myocyte hypertrophy (39). Extensive endometrial gland hyperplasia and
hypertrophy (14) and uterine vasculogenesis (56) also occur during gestation. In view of the
growth and development promoting effects of lysophospholipids (20, 32) and their role in myosin
light chain phosphorylation (10, 37), we investigated changes in the expression of receptors and
enzymes involved in Sph-1-P and LPA metabolism and signaling in the rat uterus to begin to
elucidate their role in the adaptation of the rat uterus to pregnancy.
MATERIALS AND METHODS
Materials. Progesterone was purchased from Schein Pharmaceutical (Florham Park, NJ).
Onapristone (Schering, Germany) was a gift from Dr. Robert Garfield of this Department.
Estradiol-17β, RU486, and other chemicals were purchased from Sigma Chemical (St Louis,
MO). Steroids were dissolved in sesame oil and injected subcutaneously in a total amount of 0.2
ml, except onapristone, which was dissolved in benzyl benzoate and castor oil.
Animals. Experimental protocols were approved by the Animal Care and Use Committee of the
University of Texas Medical Branch at Galveston. Uterine samples were taken from timed-
pregnant rats (Sprague-Dawley, Charles River Laboratory, Wilmington, Mass) that were
randomly selected. Pre-term delivery was induced by the administration of single dose of
onapristone (3 mg, sc) on day 17. The control group received vehicle alone. Pregnancy was
prolonged by subcutaneous administration of progesterone (2 mg, sc) twice daily, starting on
day 19. Nonpregnant rats (9 weeks old, 200-225 g) from the same vendor were treated one
week after bilateral ovariectomy with either estradiol-17β (0.2 µg), progesterone (1 mg), or both
for 4 days. Uteri were removed on the fifth day after initiation of treatment. Another group of rats
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